Leptographium Tereforme Sp. Nov. and Other Ophiostomatales Isolated from the Root-Feeding Bark Beetle Hylurgus Ligniperda in California

Home , Ophiostoma, Ophiostomatales, Sporothrix

Mycologia, 103(1), 2011, pp. 152–163. DOI: 10.3852/10-096 # 2011 by The Mycological Society of America, Lawrence, KS 66044-8897

Leptographium tereforme sp. nov. and other Ophiostomatales isolated from the root-feeding bark beetle Hylurgus ligniperda in California

Sujin Kim INTRODUCTION Thomas C. Harrington1 Hylurgus ligniperda (Fabricius) (Coleoptera: Scolyti- Department of Plant Pathology, Iowa State University, Ames, Iowa 50010-3221 dae, sensu Bright 1993, Wood 2007), a pine-infesting bark beetle, is native to Europe but has spread Jana C. Lee globally in solid wood packing material, dunnage or Horicultural Crops Research Unit, USDA ARS, logs (Haack 2006). Hylurgus ligniperda was intro- Corvallis, Oregon 97330 duced accidentally to Chile, New Zealand and South Steven J. Seybold Africa, and it recently was introduced into the USA. Chemical Ecology of Forest Insects, Pacific Southwest The first established breeding population in North Research Station, USDA Forest Service, Davis, America was discovered in 2000 in New York, California 95616 although specimens had been collected from the same general area as early as 1994. Hylurgus ligniperda also was detected in July 2003 in Los Angeles, Abstract: The redhaired pine bark beetle Hylurgus California, and is abundant and well established from ligniperda (F.) is native to Europe but was discovered northern Los Angeles and Ventura counties to San in Los Angeles, California, in 2003. This root- and Diego County in southern California (Liu et al. 2007). stump-feeding beetle is a common vector of Ophios- Hylurgus ligniperda is a root- and stump-infesting tomatales, which are potential tree pathogens or beetle whose elytral declivity is covered with reddish causes of blue stain of conifer sapwood. In this study hairs, hence its common name, the redhaired pine Ophiostomatales were isolated on a cycloheximide- bark beetle (Lee et al. 2007). This beetle has a amended medium from 118 adult H. ligniperda relatively broad host range, including pine, spruce collected from infested logs of Pinus halepensis and and Douglas-fir. It is generally considered a secondary P. pinea at two sites in California. In total eight species pest because it does not aggressively kill trees (Haack of Ophiostomatales were identified and seven species 2006), although it may become lethal when the trees that occasionally were isolated were unidentified. The are stressed or injured (Neumann 1987). The beetle most frequently isolated species were Ophiostoma ips may cause minor economic damage by introducing and Grosmannia galeiforme,whichwereisolated sapstain fungi to wood (Harrington 1988, Zhou et al. respectively from 31% and 23% of the 118 beetles. 2004a), and it could become an effective vector of a The other species isolated included O. piceae (isolated tree pathogen or blue-stain fungus (Harrington 1988, from 9% of the beetles), O. querci (8%)and 1993, 2005; Jacobs and Wingfield 2001; Paine et al. Leptographium tereforme sp. nov. (6%). Grosmannia 1997; Six 2003). huntii, L. serpens,threeSporothrix species, O. Most common blue-stain fungi on conifer sapwood floccosum, O. stenoceras, two unidentified Hyalorhino- have been classified in the genera Ophiostoma, cladiella sp. and a sterile fungus each were isolated Ceratocystiopsis, Grosmannia and Ceratocystis. These from fewer then 5% of beetles. Most of the identified genera represent two phylogenetically unrelated species already were known in USA and have been groups, Ophiostomatales, close to Diaporthales, and found in association with H. ligniperda in other Ceratocystis, in Microascales (Harrington 2005, Har- countries. However the new species, L. tereforme, and rington et al. 2010, Zipfel et al. 2006). Most blue-stain G. galeiforme were recorded from USA for the first fungi associated with conifer bark beetles belong in time, and this is the first report of L. serpens from Ophiostomatales, although a few conifer bark beetles western North America. transmit Ceratocystis species (Harrington 2005, Kirisits Key words: Grosmannia galeiforme, Grosmannia 2004). Sexual species in Ophiostomatales produce huntii, Hyalorhinocladiella spp., Leptographium ser- sticky ascospores at the tips of perithecia, and pens, Leptographium tereforme, Ophiostoma ips, Ophios- anamorphs of Ophiostomatales typically produce toma piceae, Ophiostoma querci, redhaired pine bark wet droplets of conidia. The sticky spores may be beetle, Sporothrix spp. acquired by bark beetles in mycangia or, more commonly, on the exoskeleton (Harrington 1993, Submitted 4 Apr 2010; accepted for publication 7 Jun 2010. 2005; Six 2003). Zipfel et al. (2006) redefined 1 Corresponding author. E-mail: [email protected] Ophiostoma and distinguished Ophiostoma species

152 KIM ET AL.: LEPTOGRAPHIUM TEREFORME SP. NOV. 153

(with Pesotum, Hyalorhinocladiella and Sporothrix the presence or absence of an aedeagus. Each adult was anamorphs) from Ceratocystiopsis (with Hyalorhino- killed by crushing with flame-sterilized forceps and shipped cladiella anamorphs) and Grosmannia (with Leptogra- from California to Iowa, where the beetles were refrigerated phium anamorphs). Mullineux and Hausner (2009) until processing. studied the secondary structure of the internal Each beetle was cut aseptically into four parts (head, prothorax, mesothorax and abdomen), and each part was transcribed spacer regions of rDNA and further placed in cycloheximide-streptomycin malt agar (CSMA, supported separation of Grosmannia from Ophios- 10 g malt extract, 15 g agar, 200 mg cycloheximide and toma, but there is still some question as to the 100 mg streptomycin per 1 L dH2O) (Harrington 1981, monophyly of Grosmannia (Harrington et al. 2010). 1992). Streptomycin and cycloheximide were added after Ophiostomatales have been isolated from H. autoclaving. Plates were incubated at room temperature ligniperda collected in South Africa (Zhou et al. until colonies became visible on the medium. Cultures were 2001), Chile (Zhou et al. 2004a), New Zealand (Reay transferred via conidial masses or the edge of the mycelium et al. 2006) and Spain (Romoń et al. 2007). Zhou et to fresh plates of 1.5% malt extract agar (MEA, 15 g malt al. (2001) reported eight Ophiostomatales from H. extract and 20 g agar per 1 L dH2O). Pure cultures were ligniperda that were obtained from infested stumps used for DNA extraction and morphological examination. and root collars of Pinus patula and P. elliottii in Fungal morphology.—Representative isolates of each puta- South Africa. Ophiostoma ips, L. lundbergii Lagerb. & tive species were stored at 280 C in the collection at Iowa Melin and L. serpens were commonly isolated species, State University. Isolates were grown at room temperature and Ceratocystiopsis minuta (Siemaszko) Upadh. & on 1.5% MEA and examined with light microscopy with Kendrick, G. galeiforme, O. piceae, O. stenoceras and O. differential interference contrast (DIC) microscopy (by pluriannulatum (Hedgc.) H. & P. Sydow were isolated Normarsky, Olympus BH-12 with Kodak DS 120). All major from fewer than 2% of beetles (Zhou et al. 2001). morphological characters were compared with described Reay et al. (2006) isolated respectively G. galeiforme Ophiostomatales. Ten day old cultures were incubated at 25 C on MEA. and G. huntii from 96% and 83% of adult H. Thirty measurements were made for each character from ligniperda in New Zealand; O. floccosum, O. setosum each isolate studied, and the mean and range were Uzunovic, Seifert, S.H. Kim & C. Breuil, O. ips, O. computed for each character from each isolate. Colonized querci, L. procerum (W.B. Kendr.) M.J. Wingf. and L. agar plugs (5 mm diam) were taken from 10 d cultures and truncatum (M.J. Wingf. & Marasas) M.J. Wingf. were placed in the middle of fresh MEA plates, mycelium side isolated less frequently. Zhou et al. (2004a) isolated down, for a temperature growth study. The plates were only C. minuta, G. galeiforme and O. ips from 80 H. inverted and incubated in the dark at 5, 15, 25, 30 or 35 C. ligniperda adults collected in Chile. Romoń et al. After 10 d colony radius was calculated by taking two radial (2007) isolated only L. guttulatum M.J. Wingf. & K. measurements from each of three replicate cultures and Jacobs from five H. ligniperda adults in Spain. The averaging the six measurements. fungi associated with H. ligniperda in USA have not Mating experiments.—For synnemata-forming species and been documented. some Leptographium species, representative isolates were This study explored the relationship between H. paired with tester strains of O. floccosum (C989, mat-a, and ligniperda and Ophiostomatales in California to C988, mat-b, from New Zealand), G. galeiforme (DM269-1, examine whether the beetle brought potential tree mat-a, and DM269-2, mat-b, from Scotland), G. huntii pathogens or new blue-stain fungi to North America (DM1870, mat-a, from Chile), O. piceae (C1618, mat-a, and and whether the beetle might have begun carrying C1620, mat-b, from Chile) or O. querci (C1018, mat-a, and native North American fungi. C1017, mat-b, from New Zealand). Pairings were made on pine twig media (PTM, 1.5% MEA with one piece of autoclaved twig) (Harrington 1992). Debarked twigs of MATERIALS AND METHODS Pinus strobus L. were cut 3–4 cm long and autoclaved twice for 50 min. One sterilized twig was placed aseptically in each Beetle collection and isolation.—In Mar 2006 118 adult H. Petri dish, and autoclaved 1.5% MEA was poured over the ligniperda, 55 females and 63 males, were collected live from twig until covered. In most cases two isolates were placed egg galleries in the phloem (inner bark) of cut stem next to each other on PTM and the plates were incubated at sections of pine at two sites in California. Seventy-three room temperature until perithecia appeared. In other cases adult H. ligniperda were collected from Pinus halepensis an isolate was grown on PTM until fully colonized and the Mill. and P. pinea L. at Descanso Gardens (La Canãda mycelium was exposed to spermatozoa with 20 mL conidial Flintridge, 34.21055uN, 118.20044uW), and 45 adult H. slurry of the second isolate (Harrington and McNew 1997). ligniperda were collected from P. halepensis and P. pinea at Bonelli Regional Park (San Dimas, 34.08856uN, DNA sequencing and RFLP analysis.—Each isolate was 117.81253uW). Adults were placed individually in 1.5 mL grown at room temperature 1 wk on malt yeast extract agar microcentrifuge tubes and stored on ice. Sex was deter- (MYEA, 15 g malt extract, 2 g yeast and 20 g agar per 1 L mined by dissecting the terminal end of the abdomen for dH2O), mycelium and spores were scraped from the plate, 154 MYCOLOGIA and DNA was extracted from the scrapings with PrepManTM tained Leptographium anamorphs, the third group Ultra (Applied Biosystems) following the manufacturer’s contained only Sporothrix anamorphs and the fourth protocol. group had Hyalorhinocladiella anamorphs. Another Attempts were made to amplify the 26S rDNA gene species produced no spores in culture. (nuclear large subunit, LSU) and the internal transcribed Many species were similar to common Ophiostoma spacer (ITS rDNA) regions of representative isolates of each putative species. The LSU region was amplified with primers and Leptographium species (pigmented and macro- LROR and LR5 (Vilgalys and Hester 1990). The ITS regions nematous conidiophores) reported from H. ligni- and the 5.8S gene of the ribosomal RNA operon were perda from the southern hemisphere. The most amplified with primers ITS1-F (Gardes and Bruns 1993) and common Ophiostoma species were distinguished by ITS-4 (White et al. 1990). Template DNA was amplified in a comparing rDNA sequences and morphological 50 mL single reaction volume, containing 1.25 units Takara descriptions of Ophiostomatales. Overall eight spe- Ex Taq Polymerase, 13 PCR reaction buffer, 200 mM dNTPs, cies were identified but seven could not be identified 5% (V/V) DMSO, and 0.25 mM of each primer (Takara to species. One of the unidentified species is Mirus Bio, Japan). Cycling conditions were initial denatur- described here as new. The species are reported ation at 94 C for 2 min, 35 cycles of annealing at 49 C for below in decreasing order of isolation frequency. LSU and at 54 C for ITS for 35 s, and primer extension at 72 C 2 min, followed by one final cycle of primer extension Isolated species.—Ophiostoma ips (Rumbold) Nannf. at 72 C 15 min (Harrington et al. 2001). O. ips was easily distinguished morphologically from PCR products either were purified with a QIAquick PCR Purification Kit (QIAGEN Inc., California) or were digested other fungi by brown mycelium and ascospores with with restriction enzyme. PCR products were sequenced at rectangular sheaths. Many O. ips isolates produced the DNA Synthesis and Sequencing Facility at Iowa State perithecia and ascospores in culture within 3–4 wk University with primers ITS1-F and ITS-4 for the ITS region after incubation. Perithecia dark brown to black, bases or LROR and LR3 (Vilgalys and Hester 1990) for the LSU. globose, (140–)160–400(–490) mm diam, with straight The restriction enzyme HaeII (Gibco BRL. Inc., USA), or curved necks, 180–1100(–1300) mm long, and no which recognizes the base sequence RGCGC/Y, was used to ostiolar hyphae. Ascospores oblong, surrounded by a identify members of the O. piceae complex (Harrington et hyaline gelatinous sheath, appearing rectangular or al. 2001). Each unpurified PCR product (10 mL) was mixed square, 2.4–4.8 3 1.3–2.5 mm. Conidiophores hyaline, with 2 mL103 buffer (supplied with the enzyme), 1 mL (50–)79–215(–240) mm long, conidiogenous cells restriction enzyme, and 7 mL sterilized, distilled water. Digestion was allowed to procede 3 h at 37 C, and then without denticles. Conidia cylindrical to slightly restriction fragments were separated by electrophoresis 3 h lageniform, 2.3–5.8(–7) 3 (1–)1.5–2.5(–4) mm. ITS at 70 V in 1.6% agarose gels with TBE buffer (89 mM Tris, sequence of the O. ips isolates (GU129980) matched 89 mM boric acid and 2 mM EDTA, pH 8.0). (594 of 594 nucleotides) those of O. ips isolates from different countries (e.g. AY546707, AY172021 and Comparison of rDNA sequences.—Sequences of the LSU and ITS rDNA were compared with those of isolates of DQ539549). Ophiostomatales from our database and others available Grosmannia galeiforme (Bakshi) Zipfel Z.W. de Beer in the NCBI database with MegaBLAST (BLASTN 2.2.22 +). & M.J. Winf.AllG. galeiforme isolates had light gray to dark brown mycelium and synnematous conidiophores (common) and rarely mononematous conid- RESULTS iophores. These isolates produced perithecia and Isolation of fungi from Hylurgus ligniperda.—In total ascospores on PTM 6–8 wk when paired with mating 118 H. ligniperda adults were sampled for the testers. Perithecia bases, (120–)195–240(–280) mm presence of Ophiostomatales and 114 individual diam, necks (510–)600–710 mmlong.Ascospores Ophiostomatales isolates were obtained on CSMA. reniform with sheath, 1.8–4.3 3 1.0–2.4 mm. Synne- Of the 118 beetles 87 (74%) yielded at least one mata 120–300 mm tall. Conidia cylindrical, (2–)3.2–5 species of Ophiostomatales. Twenty-two percent of 3 1–2.5 mm. ITS sequences of all G. galeiforme isolates beetles yielded more than one species of Ophiosto- (e.g. GU129981) matched (514 of 514 nucleotides) matales. On average 0.96 species of Ophiostomatales with the previously reported sequences of G. galei- were isolated from each adult sampled. forme (AY649770, DQ062979). All isolated Ophiostomatales were grouped into 15 Ophiostoma piceae (Mu¨ nch) H. & P. Sydow. All O. putative morphological species based on mycelial piceae isolates had Pesotum and Sporothrix synana- characteristics, teleomorphs, anamorphic states and morphs, and all tested isolates of O. piceae produced growth rate. Fourteen of the putative species were perithecia on PTM in 2 wk when paired with separated into four main groups based on anamor- appropriate mating type testers. Perithecia with phic states. The first group contained Pesotum and globose bases, 110–200 mm diam, and straight necks, Sporothrix synanamorphs, the second group con- 610–960 mm long. Ascospores reniform, (2–)2.5–4.5 3 KIM ET AL.: LEPTOGRAPHIUM TEREFORME SP. NOV. 155

1.3–2.1 mm, accumulating on top of the perithecial tion G. robustum forms a teleomorph in culture neck. Synnemata dark brown, (160–)210–1600(–1930) (Jacobs and Wingfield 2001, Robinson-Jeffrey and mm long. Conidia cylindrical and ovoid to oblong, (2–) Grinchenko 1964), but Leptographium sp. A formed 2.8–5(–6) 3 1.0–2.3 mm. All O. piceae isolates had the no perithecia in culture, even when isolates were same HaeII restriction pattern, with fragments of 400, paired. Leptographium sp. A is described later as a new 200, 120 bp, as had been reported for this species species. (Harrington et al. 2001). Furthermore ITS sequences Sporothrix sp. C. Isolates of this unidentified of isolates of O. piceae (e.g. GU129988) matched (575 Sporothrix formed colonies that were white to pale of 575 nucleotides), the previously reported sequences yellow on MEA. Conidiophores short, arising from thin of O. piceae (EF506934 and AF493249). septate hyphae. Conidiogenous cells with conspicuous Ophiostoma querci (George´vitch) Nannf. All O. denticles. Conidia ovoid to cylindrical with pointed querci isolates produced a nut-like aroma and had a ends, 2.1–4.5(–5.3) 3 0.5–1.2 mm. The ITS sequence concentric ring pattern when grown on MEA (Har- of this species (GU129986) was closely related to rington et al. 2001). Most isolates formed light brown other unidentified Sporothrix species (de Beer et al. protoperithecia on MEA, and some isolates produced 2003a) that were isolated from either soil or plant perithecia and ascospores on MEA by themselves material (AY484468, 531 of 536 bases matching; and within 10 d, and the perithecia had distinctive, long AF484471, 527 of 536 bases matching) and the perithecial necks with glistening ascospore droplets, recently described (de Meyer et al. 2008) S. stylites similar to O. pluriannulatum perithecia (Halmschla- (EF 127884, 527 of 536 base pairs matching and ger et al. 1994, Harrington et al. 2001). All O. querci EF127882, 530 of 536 bases matching). isolates had Pesotum and Sporothrix synanamorphs. Grosmannia huntii (Robins.-Jeff.) Zipfel Z.W. de Beer Perithecia with globose bases, 150–215 mm diam, necks & M.J. Wingf. Isolates of G. huntii failed to produce straight or curved, 710–1420 mm long, with ostiolar perithecia, even when paired with the mat-a tester of hyphae. Ascospores reniform, 1.8–4.3 3 1.0–2.4 mm, G. huntii from Chile. However the Leptographium accumulating on the tip of the neck. Synnemata dark state of the California isolates was consistent with brown to black, 290–830(980) mm long. Conidia reports for G. huntii (Jacobs et al.1998, Jacobs and cylindrical or ovoid to oblong, 3–4.8 3 1–2.8 mm. All Wingfield 2001, Robinson-Jeffrey and Grinchenko O. querci isolates had the same HaeII restriction 1964). Colonies dark greenish olivaceous with smooth pattern, with fragments of 520 and 200 bp, consistent serpentine hyphae. Conidiophores light olivaceous, with reports for this species (Harrington et al. 2001). arising singly or in groups, 110–540 mm long, with Furthermore ITS sequences of the O. querci isolates 2–3 primary branches, 5–20 3 3–6 mm. Conidia (e.g. GU129989) matched (578 of 578 nucleotides) obovoid with truncate bases and rounded apices, with the sequences of other previously reported O. (3–)4–5.4(–6) 3 1.5(–3) mm. The ITS sequences of querci isolates (EF429089, FE506936, AF493246 and California isolates (e.g. GU129982) were identical to AY328520). the ITS sequences of other G. huntii isolates, such as Leptographium sp. A. All Leptographium sp. A DQ 674362 and DQ674361 (588 of 588 bases isolates had identical ITS rDNA sequences matching). (GU129991, GU129992, GU129993, GU129994 and Leptographium serpens (Goid.) Siem. Morpholog- GU129995) that were unique but similar to those of ical characters of isolates fell within the range G. aureum (Rob.-Jeffr. & Davids.) Zipfel Z.W. de Beer reported for L. serpens (Jacobs and Wingfield 2001) & M.J. Winf. (AY935606), G. clavigerum (Rob.-Jeffr. & and matched other isolates of L. serpens in the Iowa Davids.) Zipfel Z.W. de Beer & M.J. Winf. (AY263196), State University collection (C391 and C759 5 CMW G. robustum (Rob.-Jeffr. & Davids.) Zipfel Z.W. de 290). Hyphae serpentine. Conidiophores with a large Beer & M.J. Winf. (AY263190, AY263189), L. long- central metula, 3–5 primary metulae 8–20 3 4–10 mm, iclavatum Lee, Kim & Breuil (AY816686), L. terebran- three or more secondary metulae on top of each tis Barras & Perry (AY935607) and L. pyrinum primary metula. Conidia 3–5 3 1–2 mm. L. serpens Davidson (AY544621). Of the related species, Lepto- originally was described from Pinus sylvestris L. in graphium sp. A was morphologically most similar to G. Italy and the description included a teleomorph robustum because of its globose conidia and granular (Goidanich 1936), but the only material available material on the conidiophore stipes and hyphae, from the original collection is an isolate that does not although G. robustum forms the granular material produce perithecia (Harrington 1988). Thus Har- only on hyphae (Jacobs and Wingfield 2001, Robin- rington (1988) suggested using the anamorphic name son-Jeffery and Davidson 1968). The conidiophores of L. serpens instead of O. serpens. Although it was not Leptographium sp. A are shorter than those of G. possible to obtain a full ITS sequence, the partial ITS- robustum and other Leptographium species. In addi- 2 sequence of L. serpens that was obtained was similar 156 MYCOLOGIA to accessions AY554488 and AY707203 of L. serpens with pointed end, small, 3.2–4.8(–6.2) 3 1–1.6(–2.4) (81 bases matching). The LSU sequence (GU129998) mm. In contrast Sporothrix sp. B is very similar to of L. serpens matched closely the sequences of other isolates from the southern pine beetle (C349 and previously reported isolates of L. serpens, such as C558): colonies with abundant aerial mycelia and a EU177471 (isolate C30, from the holotype) and concentric ring pattern. Conidia 4–5.6(–6.4) 3 1– AY707203 (both with 541 of 542 bases matching). 1.8(–2.5) mm with large denticles 0.2–0.4 mm. The Ophiostoma floccosum Mathiesen. O. floccosum separation of Sporothrix sp. A and B was supported by isolates from California did not form perithecia and comparison of ITS sequences. All isolates of Sporothrix ascospores when paired with testers of opposite sp. A had ITS sequences (GU129984) similar to those mating type, but they did produce typical Pesotum of western pine beetle isolates (DQ396788, 537 of 543 and Sporothrix anamorphic states. Synnemata red- bases matching and AF484452, 536 of 543 bases brown with lateral knobs, 120–280(–350) mm long, matching). ITS sequences of isolates of Sporothrix sp. secondary synnemata frequently emanating from the B (GU129985) matched those of southern pine beetle conidial masses on the top of primary synnemata. isolates C349 and C558 of O. nigrocarpum (541 of 541 Conidia cylindrical or fusiform with pointed base, 3–9 bases matching) and were similar to those of O. 3 1–2.8 mm. These fungi had the HaeII restriction abietinum Marmolejo & Butin (DQ396788 and pattern typical of the ITS product (280 and 200 bp) of AF484453, 538 of 541 bases matching). O. floccosum (Harrington et al. 2001). Hyalorhinocladiella sp. A. Colonies white, with no Ophiostoma stenoceras (Robak) Melin & Nannf. aerial mycelium. Hyphae thick-walled, submerged Colonies white to slightly yellowish, growth 85 mm under the medium. Conidiophores arising directly diam after 20–24 d at 25 C. Protoperithecia developed from hyphae, lacking prominent denticles. Conidia after 2 wk, and then perithecia and ascospores broadly ellipsoidal to subcylindrical, with rounded developed and matured slowly. Perithecia with bases ends, (0.9–)1.2–1.9 3 3.2–5.2 mm. In the DNA (70–)95–170(–210) mm diam, usually with a single sequence comparison isolates of Hyalorhinocladiella long, straight neck, 520–1500(–1800) mm, but some sp. A were similar to those of O. rectangulosporium with two short necks, (280–)300–420 mm, ostiolar based on ITS (GU129997, 528 of 549 bases matching hyphae present. Ascospores hyaline and curved or DQ539538) and LSU (GU221905, 518 of 538 bases crescent-shaped, small, 2–3(–3.9) 3 1–1.5 mm. Conid- matching AB235158). iophores with distinct denticles on the conidiogenous Hyalorhinocladiella sp. B. Conidiophores with large cells. Conidia elongated or ellipsoidal with pointed conidial masses. Hyphae hyaline, thick-walled, sub- ends, hyaline, (2–)2.8–6.5(–7.5) 3 (1–)1.3–2 mm. The merged in the medium. Condiophores short, lacking ITS sequence of the O. stenoceras isolates (GU129990) distinct denticles at the point of conidium detach- matched (538 of 539 bases) sequences of other O. ment. Conidia broadly ellipsoidal with both ends stenoceras isolates, including accessions DQ539511, rounded, (7.5–)13–18 3 4.5(–5) mm. Isolates of AY280492 and AF484476. Hyalorhinocladiella sp. B had ITS sequences Sterile fungus. This species did not produce peri- (GU129996) close to those of O. piliferum (Fries) H. thecia or conidia on MEA, MYEA or PTM. Colonies & P. Sydow (AY934516, 444 of 481 bases matching), white and fast-growing, filling an 85 mm plate within a O. bicolor Davids. & Wells. (DQ268606, 442 of 481 week at 25 C. Ophiostoma species without teleomorph bases matching), and O. montium (Rumbold) von or anamorph in culture are unusual, although Ohtaka Arx. (AY546710, 454 of 481 bases matching) and LSU et al. (2006) reported that O. rectangulosporium from sequences (GU221906) close to those of O. bicolor Japan had a teleomorph state but not an anamorph (DQ268605, 529 of 539 bases matching) and Hya- state in culture. The sterile species had an ITS lorhinocladiella sp. (DQ268591, 526 of 539 bases sequence (GU129987) that was similar to sequences matching). of O. rectangulosporium (AY242825, 532 of 550 bases matching and GU124171, 529 of 550 bases matching). Sporothrix species A and B (near O. nirgrocarpum TAXONOMY [Davids] de Hoog). These two unidentified species Leptographium tereforme S.J. Kim & T.C. Harrin. sp. differ slightly in their morphological characters. nov. FIG.1 Sporothrix sp. A is similar to O. nigrocarpum isolates MycoBank MB518036 from the western pine beetle (C190, C210): Colonies Coloniae olivacea vel atro-viridae, ad 34 mm in 10 dies in 21 grow slowly on MEA, 2.3–2.7 mm d at 25 C, white to 1.5% MEA. Hyphae brunnea, granulatae. Conidiophorae light yellowish, aerial mycelia with no concentric ring singulae aut aggregatae, (35–)70(–130) mm longae. Stipae pattern. Conidiogenous cells usually short and slightly brunnea vel atro-brunnea, granulatae, 1–4-septatae, (13–) rough, with distinct denticles. Conidia clavate to ovoid 20–60(–65) mm longae et (2–)3–6(–8) mm latae, con 0–5 KIM ET AL.: LEPTOGRAPHIUM TEREFORME SP. NOV. 157

FIG. 1. Condiophores, conidia and hyphae of Leptographium tereforme. A–C. Conidiophores with stipes covered with brown, granular material. A, C. Conidiophores with primary and secondary metulae. B. Conidiophore with no metulae. D. Hyphae covered with granular material. E. Conidia. F–I. Conidiogenous cells showing holoblastic and enteroblastic conidium formation. Bars: A–C 5 20 mm, D–I 5 10 mm. metulae primae. Conidia hyalinae, eseptatae, oblongatae vel green or dark gray with age. No growth at 5 C or 35 C. obovatae, apicibus rotundatis, 3.2–5(–8) 3 2.5–3.9(–4.8) mm. Hyphae mostly submerged, covered by granular Specimens examined. USA. CALIFORNIA: Los Angeles material (FIG. 1D). Perithecia and ascospores absent. County, La Canãda Flintridge, Descanso Gardens, Conidiophores occurring singly or in groups (3–4), 34.21055uN, 118.20044uW, from female Hylurgus ligniperda, micronematous, mononematous, (35–)70(–130) mm 26 Mar 2006, S.J. Kim, C2314 (Holotype, BPI879603, a long (FIG. 1A, B). Rhizoid-like structures absent. Stipe dried culture of isolate C2314 5 CBS125736). brown to dark brown, becoming darker, covered with Etymology. Latin teres (rounded) and forme (form, granular material on lower half to lower three- shape), referring to the rounded conidia. quarters, 1–4-septate, (13–)20–60(–65) mmlong Additional cultures examined. USA. CALIFORNIA: Los (from base to below primary branches), (2–)3–6 Angeles County, La Canãda Flintridge, Descanso Gardens, (–7.5) mm wide below primary branches, apical cell from female H. ligniperda, 26 Mar 2006, S.J. Kim, C2409; Los Angeles County, La Canãda Flintridge, Descanso not swollen, (3–)4–7(–10) mm wide at base, basal cell Gardens, from male H. ligniperda, 26 Mar 2006, S.J. Kim, not swollen. Conidiogenous apparatus (15–)21–37 C2421; Los Angeles County, La Canãda Flintridge, Des- (–42) mm long excluding the conidial mass, with 1–4 canso Gardens, from male H. ligniperda, 26 Mar 2006, S.J. series of cylindrical branches (FIG.1C). Primary Kim, C2422; Los Angeles County, La Canãda Flintridge, branches, if present, hyaline, smooth, cylindrical, Descanso Gardens, from female H. ligniperda, 26 Mar 2006, (8–)9–18(–30) mm long. Secondary branches absent S.J. Kim, C2426. or present, hyaline, (5–)10(–15) mm in long. Conidia Colonies with optimal growth at 25 C on 1.5% MEA, hyaline, aseptate, oblong to slightly obovoid with attaining 35 mm diam in 10 d, olive green to dark round apices, 3–5(–8) 3 2.5–4.0(–5) mm(FIG. 1E). 158 MYCOLOGIA

TABLE I. Number of successful isolations and frequency (percent, in parentheses) of Ophiostoma and related species from adult Hylurgus ligniperda from New Zealand, La Can˜ada Flintridge, and San Dimas, California and representative ITS and LSU rDNA sequences

California, USA GenBank accession number3 New Zealand1 La Can˜ada Flintridge San Dimas Species (n 5 120)2 (n 5 73) (n 5 45) ITS LSU Grosmannia galeiforme 115 (95.8%) 18 (24.6%) 9 (20%) GU129981 GU221908 Ophiostoma ips 6(5%) 8 (11%) 28 (62%) GU129980 Ophiostoma piceae 0 11 (15%) 0 GU129988 Ophiostoma querci 2 (1.7%) 10 (13.7%) 0 GU129989 Leptographium tereforme 0 7 (9.6%) 0 GU129994 Grosmannia huntii 100 (83.3%) 2 (2.7%) 3 (6.7%) GU129982 GU129999 Sporothrix sp. C 0 4 (5.5%) 1 (2.2%) GU129986 Leptographium serpens 0 2 (2.7%) 2 (4.4%) GU129983 GU129998 Ophiostoma floccosum 6(5%) 2 (2.7%)0 Sporothrix sp. A 0 1 (1.4%) 1 (2.2%) GU129984 GU221907 Ophiostoma stenoceras 0 1 (1.4%) 0 GU129990 Sporothrix sp. B 0 1 (1.4%) 0 GU129985 Hyalorhinocladiella sp. A 0 1 (1.4%) 0 GU129997 GU221905 Hyalorhinocladiella sp. B 0 1 (1.4%) 0 GU129996 GU221906 Sterile fungus 0 1 (1.4%) 0 GU129987 Ophiostoma setosum 7 (5.8%)0 0 Leptographium procerum 7 (5.8%)0 0 Leptographium truncatum 1 (0.8%)0 0 No fungus isolated Not available 24 (32.9%) 8 (17.8%)

1 Data from Reay et al. 2006. 2 Number of adult beetles sampled. 3 GenBank accession numbers correspond to sequences of representative isolates of each species.

Conidial droplet hyaline at first, becoming creamy fungus were found only at La Can˜ada Flintridge yellow, remaining the same color when dry. (TABLE I).

Isolation frequencies.—Ophiostoma ips was isolated from 31% of 118 beetles sampled. In addition to O. DISCUSSION ips other fungi with high frequencies of isolation Fifteen Ophiostomatales species were isolated from included G. galeiforme (from 23%), O. piceae (9%), O. the root-feeding bark beetle Hylurgus ligniperda in querci (8%)andL. tereforme (6%). In contrast California. Most of the previously isolated species had Sporothrix sp. C, G. huntii, L. serpens, O. floccosum been reported from USA, mostly as blue-stain fungi on and Sporothrix sp. A each were isolated from fewer pine sapwood (Jacobs and Wingfield 2001, Harrington than 5% of beetles (TABLE I). Each of the other 1988, Harrington et al. 2001, Upadhyay 1981). species, including O. stenoceras, were isolated only Ophiostoma ips, O. piceae, O. querci, O. floccosum and from one beetle. O. stenoceras are commonly isolated species from H. No difference in frequency of isolation was ob- ligniperda and have been reported from other bark served between male vs. female beetles at either site beetles in North America and worldwide (Harrington for any of the fungal species based on a Chi-square et al. 2001, Reay et al. 2006, Romoń et al. 2007, Zhou et analysis (P 5 0.05). However there were differences in al. 2001, 2004a). Fungi associated with H. ligniperda on frequency of isolation of some species between the other continents but not found in western North two sites. At San Dimas O. ips was the dominant America included G. galeiforme and L. serpens,soitis species with a 62% frequency of occurrence from the possible that these two species were introduced to 45 sampled beetles and five different fungal species California with the beetle. were isolated. At La Canãda Flintridge O. ips was less Of all the species isolated in this study G. galeiforme common (from 11% of beetles) and G. galeiforme was was isolated consistently from H. ligniperda at the dominant species with 25% frequency of occur- both California sites. This species is believed to be rence, and O. piceae and O. querci were frequently European (Bakshi 1951, Mathiesen-Kaä¨rik 1960) and isolated. In addition L. tereforme and the sterile has been associated with a wide range of European KIM ET AL.: LEPTOGRAPHIUM TEREFORME SP. NOV. 159 bark beetles, including Orthotomicus erosus (Wollas- iclavatum, L. terebrantis and L. pyrinum, by rDNA ton), Hylastes angustatus (Herbst), Dryocoetes auto- sequence comparisons and morphology. The rDNA graphus (Ratzeburg) and Ips sexdentatus (Boerner) sequences of L. tereforme were closest to those of L. (de Beer et al. 2003b, Romoń et al. 2007, Zhou et al. terebrantis, L. wingfieldii Morelet, L. truncatum (M.J. 2004b, 2004c). Grosmannia galeiforme was introduced Wingf. & Marasas) Wingfield, L. lundbergii and L. into the southern hemisphere with one or more of guttulatum M.J. Wingf. & Jacobs. Leptographium the above European bark beetles. For instance in tereforme is easily distinguished morphologically from Chile and New Zealand G. galeiforme was isolated these species by its globose conidia, short conidio- from both H. ligniperda and Hylastes ater (Paykull) phores and granular material covering half or up to (Reay et al. 2002, 2005, 2006; Zhou et al. 2004a) and three-quarters of the conidiophore stipe. L. tereforme in South Africa it was isolated from H. ligniperda is not known elsewhere, but it is possible that reports (Zhou et al. 2001). Species closely related to G. of L. guttulatum, L. lundbergii or L. truncatum from galeiforme have been reported from North America H. ligniperda in Spain, South Africa and New Zealand (Zhou et al. 2004b, c), but G. galeiforme had not been (Romoń et al. 2007, Zhou et al. 2001, 2004a) were confirmed. Thus it is likely that H. ligniperda brought actually L. tereforme. G. galeiforme with it when it was accidentally intro- Ophiostoma ips is known worldwide as a blue-stain duced to California. To date, pathogenicity of G. fungus and associated with many conifer-feeding bark galeiforme to pine has not been reported, so the beetles (Reay et al. 2002, 2005, 2006; Romoń et al. finding of this fungus in California might not be 2007; Zhou et al. 2001, 2004a, b), especially in cause for concern. association with Ips spp. on pines in North America Leptographium serpens has been isolated from (Mathre 1964, Romoń et al. 2007, Rumbold 1931, European root-feeding bark beetles, including H. Seifert 1993). The fungus was taken to several angustatus and H. ligniperda and Hylastes spp., in southern hemisphere countries through the acciden- South Africa and elsewhere (Harrington 1988, Jacobs tal introduction of various coniferous bark beetles and Wingfield 2001, Reay et al. 2002, 2005, 2006; (Zhou et al. 2001, 2004a, 2006). These include the Thwaites et al. 2005; Wingfield et al. 1988; Zhou et al. stem-infesting Ips grandicollis (Eichh.), which is native 2001, 2002, 2004a). For example 45% of Ophiosto- to North and Central America, and which has been matales isolates from H. angustatus in South Africa introduced into Australia (Stone and Simpson 1990). were L. serpens and 21% of H. ligniperda isolates were Ophiostoma ips was reported from New Zealand on H. L. serpens (Zhou et al. 2001). Leptographium serpens ligniperda, but it was isolated from only 5% of beetles has been associated with roots of declining Pinus spp. sampled (Reay et al. 2006) and it was not reported in in southeastern USA (Eckhardt et al. 2007). Lepto- isolations from five adult H. ligniperda in Spain graphium serpens has not been reported from western (Romoń et al. 2007). In South Africa it was isolated North America, but it was isolated from H. ligniperda from 13% of 199 sampled H. ligniperda adults (Zhou at both sites in California, although at low frequency. et al. 2001). In contrast this species was reported with Because L. serpens had not been reported from a high frequency of occurrence in isolations from H. western North America but it has been commonly ligniperda in Chile (Zhou et al. 2004a). At La Canãda associated with European bark beetle species such as Flintridge O. ips was isolated from only 11% of H. ligniperda it is likely that this beetle brought the beetles, although O. ips was isolated from 62% of fungus with it to California. Leptographium serpens has beetles sampled from San Dimas. The high frequency not been considered a serious pathogen of Pinus spp. of occurrence of O. ips at the latter site might have (Wingfield et al. 1988, Zhou et al. 2002) or as a been the cause of the relatively low incidence of other contributor to loblolly pine decline (Eckhardt et al. Ophiostomatales and the lower number of species 2007) in southeastern USA, and L. serpens was only isolated. Because O. ips is well known in North weakly pathogenic to wounded loblolly pine seedlings America and it appears to be inconsistently associated under low soil moisture conditions (Matustick et al. with H. ligniperda the beetle might have acquired O. 2008). However the presence of L. serpens in ips in California. California needs to be monitored, and further Ophiostoma piceae and O. querci were isolated pathogenicity tests might be warranted. frequently from H. ligniperda collected from La The new species, Leptographium tereforme,was Canãda Flintridge, and O. floccosum was isolated from isolated from 6% of H. ligniperda but only at La twobeetlesatthissite.Thesesynnema-forming Canãda Flintridge; thus H. ligniperda might have Ophiostoma species were not isolated from beetles acquired the fungus from another bark beetle. This collected at San Dimas. These species are not fungus is distinguished from related species, such as considered pathogens and are associated with blue- G. aureum, G. clavigerum, G. robustum, L. long- stain of conifers, although O. querci occurs primarily 160 MYCOLOGIA on hardwoods (Halmschlager et al. 1994, Harrington unidentified species from H. ligniperda in New et al. 2001). Ophiostoma floccosum originally was Zealand (Reay et al. 2006), Spain (Romoń et al. described from Sweden, and it has been reported 2007) or South Africa (Zhou et al. 2001). from Europe, Korea, New Zealand and North A sterile fungus from H. ligniperda in California was America (de Beer et al. 2003b, Harrington et al. placed somewhat near O. rectangulosporium, based on 2001) and has been associated with several bark rDNA sequences. Like the unidentified sterile fungus, beetle species, including H. ligniperda (Kirisits 2004, O. rectangulosporium does not form conidia in culture Reay et al. 2006, Romoń et al. 2007, Zhou et al. 2006). (Ohtaka et al. 2006) but it has been associated with In the past the synnema-forming Ophiostoma species bark beetles, such as Cryphalus montanus Nobuchi, have been difficult to differentiate, and O. querci and Polygraphus proximus Blandford, and Dryocoetes stria- O. floccosum often were treated as synonyms of O. tus Eggers, infesting Abies species (Yamaoka et al. piceae (Harrington et al. 2001); thus former studies 2004). Romoń et al. (2007) reported O. rectangulos- with H. ligniperda might have considered O. querci porium from H. ligniperda in Spain, but the rDNA and O. floccosum isolates as O. piceae. The association sequence of the Spanish isolates was not similar to of O. piceae and O. querci with H. ligniperda was those of the Japanese isolates. reported from South Africa (Zhou et al. 2001, 2006). A number of species were isolated from H. No synnema-forming species was found associated ligniperda in other studies but were not identified with H. ligniperda in Chile (Zhou et al. 2004a). In among the California isolations. Leptographium gut- Spain Romoń et al. (2007) isolated O. piceae and O. tulatum has been associated with bark beetles in the querci fromseveralbarkbeetlespecies,suchas genera Dryocoetes, Hylastes, Hylurgops and Tomicus Hylurgops palliatus (Gyllenhal), H. attenuates Erich- (Jacobs et al. 2001, Romoń et al. 2007, Wingfield and son and Tomicus piniperda (L.), but not from H. Gibbs 1991). Leptographium truncatum was isolated ligniperda. In the New Zealand study (Reay et al. rarely from H. ligniperda in New Zealand (Reay et al. 2006) synnema-forming species were distinguished 2006). Leptographium lundbergii was reported from and O. querci, O. floccosum and O. setosum were South Africa (Zhou et al. 2001), but the taxonomy of identified from H. ligniperda. However these species this species and L. truncatum had been confused are not strict associates of H. ligniperda and often are (Jacobs and Wingfield 2001, Jacobs et al. 2005, encountered in the absence of bark beetle activity Strydom et al. 1997, Zambino and Harrington 1992) (Harrington et al. 2001; Reay et al. 2002, 2006; and it is possible that Zhou et al. (2001) might have Thwaites et al. 2005). misidentified L. truncatum or L. tereforme isolates as Grosmannia huntii first was described from pine L. lundbergii. It also is possible that some fungi infested with a Dendroctonus sp. in Canada (Robinson- present on H. ligniperda in California were not Jeffrey and Grinchenko 1964). Grosmannia huntii has isolated because the beetles were killed before been confused with G. piceaperdum (Rumbold) Goid., shipping to Iowa. However this beetle is not known but Jacobs et al. (1998) separated the species by to have a mycangium, and spores of Ophiostomatales morphology and mating system. Grosmannia huntii on the exoskeleton of the beetle probably would has been associated with many bark beetle species on survive in high numbers at low temperatures for a few Pinus and Picea spp.; beetle species include D. days. Isolation frequencies of O. novo-ulmi Brasier ponderosae Hopkins, Ips pini (Say), and Hylastes macer from killed Scolytus schevyrewi Semenov that were (LeConte) in the USA (Harrington 1988, Jacobs and shipped from Colorado to Iowa were just as high as Wingfield 2001), Tomicus piniperda (L.) in Europe from beetles that were plated immediately after (Gibbs and Inman 1991) and H. ligniperda in New killing (Jacobi et al. 2007). Zealand, where G. huntii was isolated from 83% of H. Our results showed that a relatively large number of ligniperda adults (Reay et al. 2006); thus the associ- Ophiostoma species are associated with H. ligniperda ation between G. huntii and H. ligniperda found in in California. Most of the species isolated are this study was not surprising but the frequency of this common bark beetle associates in USA and around species from California beetles was low. the world, and many of the species have been Other species isolated from H. ligniperda in Califor- associated with H. ligniperda in the southern hemi- nia were from only one or two beetles. Ophiostoma sphere. It is likely that the beetle brought only a few stenoceras has been isolated frequently from several species, perhaps only G. galeiforme and L. serpens, with bark beetle species (Kirisits 2004, Romoń et al. 2007, it when introduced to California and the other fungi Zhou et al. 2001, 2006), but it is not generally were acquired through exchange of Ophiostomatales considered to be a strict associate of bark beetles. associated with cohabiting native bark beetles. Sporothrix sp. 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